Microtubule inhibitors, such as taxanes, represent one of the most widely used treatments for cancer. By perturbing the mitotic spindle, taxanes activate the spindle assembly checkpoint (SAC), which subsequently induces mitotic arrest and cell death. However, cancers vary widely in their sensitivity to taxanes, perhaps due to differences in the strength of the checkpoint and efficiency of mitotic arrest. Because the only known function of the SAC is to inhibit the ubiquitin ligase activity of the Anaphase-Promoting Complex/Cyclosome (APC), direct APC inhibitors might be more effective at inducing mitotic arrest, without toxicities associated with microtubule perturbation. Using phenotypic screens in Xenopus cell cycle extracts, combined with biochemical studies to identify targets of active molecules, we have identified two small molecules that inhibit APC dependent proteolysis. This grant application encompasses four major goals. First, to understand, at the atomic level, how TAME and cyclinal interact with their protein targets. Second, to understand the detailed mechanisms by which these compounds perturb APC activity. Third, to understand how these compounds perturb regulation of APC by the SAC. Fourth, to leverage our understanding of the cellular effects of these molecules to identify new classes of APC inhibitors. Through our studies, we hope to establish a definitive mechanistic understanding of how these compounds inhibit APC activity and lay the intellectual and technical groundwork for development of APC inhibitors as drugs to treat cancer.